Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping

To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1–2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks

Interferon-beta therapy downregulates the anti-apoptosis protein FLIP in T cells from patients with multiple sclerosis

Interferon-beta reduces clinical exacerbations in multiple sclerosis (MS) through several immunomodulatory mechanisms that may involve augmentation of programmed cell death (apoptosis) of T lymphocytes. The anti-apoptosis protein FLIP (Fas-associated death domain-like interleukin-1 beta -converting enzyme inhibitory protein) has been recently identified as a potent regulator of T lymphocyte susceptibility to apoptosis. In a prospective study, we evaluated the expression of FLIP and other apoptosis regulatory proteins in ex vivo activated T lymphocytes from MS patients, before and serially after treatment with interferon-beta. We also investigated the long-term effects of interferon-beta on T cell apoptosis in a cross-sectional study of MS patients receiving chronic drug therapy. Treatment with interferon-beta reduced the expression of FLIP isoforms in activated T lymphocytes. This reduced expression correlated with augmented T cell susceptibility to apoptosis and with clinical response to treatment. In contrast, interferon-beta therapy did not alter cellular expression of the anti-apoptotic protein Bcl-2. This downregulatory effect of interferon-beta on cellular FLIP expression was maintained following long-term therapy. Our findings suggest that interferon-beta therapy exerts a regulatory effect on peripheral T lymphocytes through a pro-apoptosis mechanism that involves the downregulation of cellular FLIP expression. (C) 2001 Elsevier Science B.V. All rights reserved

Expression of ß(2) adrenoreceptors on peripheral blood mononuclear cells in patients with primary and secondary progressive multiple sclerosis: a longitudinal six month study

Background: ß(2) Adrenoreceptor expression on peripheral blood mononuclear cells is increased in progressive multiple sclerosis. This increase has been correlated with disease activity in relapsing-remitting multiple sclerosis. Objective: To determine the ß(2) adrenoreceptor expression in primary and secondary progressive multiple sclerosis in relation to findings on magnetic resonance imaging (MRI) and clinical disease activity. Methods: 10 patients with multiple sclerosis were studied (five with primary progressive and five with secondary progressive forms of the disease) over a period of six months. Monthly clinical and MRI assessments of the brain and spinal cord were carried out. ß(2) Adrenoreceptor expression was assessed monthly using a ligand binding assay with [(125)I]iodocyanopindolol. Expression of ß(2) adrenoceptors on peripheral blood mononuclear cells was also assessed in five normal controls over a similar period. Results: The mean (SEM) value of ß(2) adrenoreceptor density for the five normal controls was 1346 (183) sites/cell, with affinity Kd of 120 (40) pM. MRI disease activity in primary progressive multiple sclerosis was reported on two occasions and on those occasions the expression of ß(2) adrenoreceptors was increased in excess of 1900 sites/cell; in the remaining 28 observations ß(2) adrenoreceptor expression was within the normal range (800 to 1900 sites/cell). In patients with secondary progressive disease, MRI disease activity was observed on 16 occasions. In these patients expression of ß(2) adrenoreceptors was increased in excess of 2000 sites/cell in all measurements except in one subject who did not show MRI activity throughout the six months period of study. The affinity of the receptors was within the normal range in all cases. Conclusions: Increased expression of ß(2) adrenoreceptors was correlated with MRI disease activity in two patients with primary progressive multiple sclerosis. In secondary progressive multiple sclerosis, increased expression of ß(2) adrenoreceptors tended not to correlate with MRI disease activity. This may reflect a persistent Th1 immune reaction in the secondary progressive form of the disease

Physiological assessment of aspects of autonomic function in patients with secondary progressive multiple sclerosis

A detailed non-invasive study of systemic and regional haemodynamic responses to a range of autonomic tests which assess sympathetic and parasympathetic pathways (mental arithmetic, cutaneous cold, isometric exercise, deep breathing, Valsalva manoeuvre and head-up tilt) were performed in ten patients with secondary progressive multiple sclerosis and ten age- and sex-matched healthy normal subjects (controls). Blood pressure rose in controls during the pressor tests and was maintained during tilt. In six out of ten patients with multiple sclerosis blood pressure was unchanged during one or more of the three pressor tests, but was maintained in all during tilt. In the controls, superior mesenteric artery blood flow fell during pressor tests and head-up tilt. In multiple sclerosis patients, superior mesenteric artery blood flow did not change during pressor tests but fell during tilt. Cardiac index rose during isometric exercise and fell during head-up tilt in controls. Forearm blood flow rose during mental arithmetic in the controls only, but fell during tilt in both groups. Individual analysis indicated that of the ten multiple sclerosis patients, four had responses during the pressor tests similar to controls. Responses to deep breathing and to the Valsava manoeuvre in controls and multiple sclerosis patients were similar. We conclude that some patients with an aggressive and disabling form of multiple sclerosis have selective autonomic dysfunction, in particular involving pressor responses, despite the lack of postural hypotension. The autonomic abnormality is likely to involve central autonomic interconnections rather than afferent or sympathetic efferent pathways. Further clarification of the nature, site and progression of these lesions is needed. Detection of these abnormalities, which may be clinically silent, may help in the prognostic and diagnostic evaluation of patients with multiple sclerosis.</p

β‐Adrenoceptor expression on circulating mononuclear cells of idiopathic Parkinson's disease and autonomic failure patients before and after reduction of central sympathetic outflow by clonidine

There is a short-term up-regulation of β-adrenoceptors on peripheral blood mononuclear cells (PBMC) after reduction of central sympathetic outflow by clonidine in normal individuals. We have studied β-adreno-ceptor number and affinity on PBMC in idiopathic Parkinson's disease (PD), pure autonomic failure (PAF), and multiple system atrophy (MSA; Shy-Drager syndrome) patients and age- and sex-matched normal controls (NC) before and after intravenous administration of clonidine, an α2-adrenoceptor agonist which lowers blood pressure predominantly by reducing CNS sympathetic outflow. Basal β-adrenoceptor density was high in PAF but within the normal range in PD and MSA patients. After clonidine there was a decrease in plasma levels of noradrenaline (NA) and adrenaline (Ad) in PD, MSA, and NC, and an increase in growth hormone (GH) in PD, PAF, and NC. In PAF, NA and Ad remained unchanged. In MSA, there was no increase in GH levels. There was an up-regulation of β-adrenoceptors on PBMC at 30 and 60 minutes after clonidine administration, which returned to baseline values after 2 hours, and the affinity of the receptors was decreased in NC and PD patients. Intracellular production of cAMP after isoproterenol stimulation demonstrated that the up-regulation was not functional. Up-regulation after clonidine did not occur in PAF and MSA patients. The observed correlation of plasma NA and sympathetic defect with basal and clonidine-induced up-regulation of β-adrenoceptors on PBMC may provide insight into β-adrenoceptor changes in other tissues and also help in differentiating subgroups of autonomic failure patients.</jats:p

Expression of βadrenoceptors on circulating mononuclear cells in hypertensives and normotensives before and after reduction of central sympathetic outflow by clonidine

We have studied βadrenoceptor number and affinity on peripheral blood mononuclear cells (PBMCs) in normotensives (NT) and hypertensives (HT), before and after intravenous administration of clonidine, an α2-adrenoceptor agonist which lowers blood pressure predominantly by reducing central nervous system sympathetic outflow. After clonidine, there was a decrease in blood pressure and plasma noradrenaline (NA) and adrenaline (Ad) levels, with an increase in growth hormone (GH) levels, in both NT and HT. There was no difference in basal βadrenoceptor densities on PBMCs between NT and HT. After clonidine at 30 and 60 min, there was an increase in βadrenoceptor density associated with a low affinity in NT. In HT, no changes were observed. The increased βadrenoceptor densities on PBMCs in NT after clonidine, returned to baseline values after 2h. Short term up-regulation of βadrenoceptors on PBMCs in NT after clonidine is accompanied by a fall in blood pressure (BP) and plasma levels of catecholamines. The changes may represent a compensatory mechanism reflecting a rapid externalization-activation of adrenoceptors residing on the internal surface of the membranes with a change of the coupling ability between the receptor and the catalytic component. In HT, although the haemodynamic and neurohormonal response to clonidine was similar to NT, short term up-regulation of receptors did not occur. The lack of such response may mirror a form of regulatory dysfunction of βadrenoceptors in HT.</p

Expression of βadrenoceptors on circulating mononuclear cells in hypertensives and normotensives before and after reduction of central sympathetic outflow by clonidine

We have studied βadrenoceptor number and affinity on peripheral blood mononuclear cells (PBMCs) in normotensives (NT) and hypertensives (HT), before and after intravenous administration of clonidine, an α2-adrenoceptor agonist which lowers blood pressure predominantly by reducing central nervous system sympathetic outflow. After clonidine, there was a decrease in blood pressure and plasma noradrenaline (NA) and adrenaline (Ad) levels, with an increase in growth hormone (GH) levels, in both NT and HT. There was no difference in basal βadrenoceptor densities on PBMCs between NT and HT. After clonidine at 30 and 60 min, there was an increase in βadrenoceptor density associated with a low affinity in NT. In HT, no changes were observed. The increased βadrenoceptor densities on PBMCs in NT after clonidine, returned to baseline values after 2h. Short term up-regulation of βadrenoceptors on PBMCs in NT after clonidine is accompanied by a fall in blood pressure (BP) and plasma levels of catecholamines. The changes may represent a compensatory mechanism reflecting a rapid externalization-activation of adrenoceptors residing on the internal surface of the membranes with a change of the coupling ability between the receptor and the catalytic component. In HT, although the haemodynamic and neurohormonal response to clonidine was similar to NT, short term up-regulation of receptors did not occur. The lack of such response may mirror a form of regulatory dysfunction of βadrenoceptors in HT.</p